Our long-term goal is to determine the molecular mechanisms underlying the diminished performance ofmuscle with age. Our overall hypothesis is that the age-related decline in muscle performance is due to cellular and molecular changes in key muscle proteins. The focus of this proposal is myosin, the major contractile protein in muscle. Our hypothesis is that the decline in force-generatingcapacity with age is due to specificstructural alterations in myosin, resulting from accumulation of post-translational modifications. We will test this hypothesis by performing physiological, biophysical and biochemical investigations of myosin from rats of different ages. The following aims will be pursued using hindlimb muscles of rats aged 10, 22, 24, 26, and 28 months. AIM 1. Determine age-related differences in single fiber force-generating capabilities. The force- generating capacity of single fibers will be determined using a permeabilized fiber preparation. A sensitive microgel technique will be used to identify myosin heavy chain isoform content of the fibers to determine muscle fiber type. We hypothesize that there will be a decline in the force-generating capacity of individualtype I and type II fibers with age. AIM 2. Use site-directed EPR spectroscopy to determine age-related changes in myosin structure. Spin labels will be attached to a selected site on the myosin head within muscle fibers and then EPR will be used to detect age-related changes in myosin structure during contraction. The advantage of EPR is its high resolution, which can detect and quantitate the distribution of structural states corresponding to the weak-and strong-binding intermediates of myosin. We hypothesize that the age-related decline in force generation is a result of a decrease in the strong-bindingstructural state of myosin. AIM 3. Determine age- related post-translational chemical modifications in myosin. Levels of nitrotyrosine, sulfhydryls, and carbonyls in myosin will be determined. We hypothesize that there are increases in post-translational chemical modifications in myosin causing alterations in myosin protein structure resulting in decreased forceproduction. The results will be invaluable for understanding the molecular mechanisms of cellular aging.